本文關(guān)鍵詞：利用維甲酸誘導(dǎo)及Wnt5a基因敲除兩種頜面發(fā)育畸形模型研究腭裂及舌發(fā)育異常的分子調(diào)控機(jī)制及相關(guān)性　出處：《大連醫(yī)科大學(xué)》2012年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 維甲酸 信號(hào)通路 頜面部發(fā)育畸形 動(dòng)物模型 基因芯片

【摘要】：以唇腭裂為代表的頜面部發(fā)育畸形發(fā)病率位于先天性發(fā)育畸形的首位。作為多基因易感性疾病，腭裂是由多重環(huán)境因素與多個(gè)基因交互作用所導(dǎo)致，因此病因復(fù)雜，機(jī)制不明。一直以來(lái)，大多數(shù)的腭裂病因?qū)W研究者均把目光集中在腭突自身的發(fā)育異常，然而，腭裂發(fā)生的原因，，一是由于腭突內(nèi)部的缺陷，二是繼發(fā)于頜面部其他組織的缺陷，如臨床上頜發(fā)育不足和下頜后縮，導(dǎo)致舌位置異常，從而物理性的阻礙腭突上抬而導(dǎo)致腭裂。但目前頜面部其他組織器官，尤其是作為與腭組織結(jié)構(gòu)、生理功能以及生長(zhǎng)發(fā)育密切相關(guān)的舌發(fā)育分子調(diào)控機(jī)制、及其發(fā)育異常與腭裂發(fā)生的關(guān)聯(lián)均少見報(bào)道。 維甲酸（Retinoic acid，RA）是維生素A的衍生物，作為化妝品添加劑以及治療皮膚病、癌癥等疾病的藥物，成為較常見的致畸的環(huán)境因素。同時(shí)維甲酸通路與WNT及SHH等信號(hào)分子通路相互作用共同參與頜面部發(fā)育的調(diào)控。本研究利用維甲酸誘導(dǎo)及Wnt5a基因敲除兩種頜面發(fā)育畸形動(dòng)物模型，通過(guò)體內(nèi)和體外實(shí)驗(yàn)，探究腭裂以及舌發(fā)育異常的分子調(diào)控機(jī)制以及二者的相關(guān)性。以期加深對(duì)舌發(fā)育的分子機(jī)制的了解，并為腭裂及舌發(fā)育異常的預(yù)防以及臨床治療提供可能的科學(xué)依據(jù)。 目的：利用維甲酸誘導(dǎo)及Wnt5a基因敲除小鼠兩種頜面部發(fā)育畸形動(dòng)物模型，研究腭裂以及舌發(fā)育異常分子調(diào)控機(jī)制以及二者的相關(guān)性。 方法：建立和應(yīng)用維甲酸誘導(dǎo)及Wnt5a基因敲除兩種小鼠頜面部發(fā)育畸形模型，體視顯微鏡及組織染色觀察表型的組織形態(tài)學(xué)變化，BrdU摻入法檢測(cè)細(xì)胞增殖，TUNEL染色方法檢測(cè)細(xì)胞凋亡，Masson染色檢測(cè)舌肌纖維化，基因芯片篩選腭裂和舌發(fā)育異常的差異基因，實(shí)時(shí)定量PCR驗(yàn)證基因芯片結(jié)果，免疫組織化學(xué)實(shí)驗(yàn)進(jìn)一步探查WNT信號(hào)分子通路GSK3β蛋白以及成肌調(diào)節(jié)因子家族成員Myf5和MyoD在舌肌中的表達(dá)變化。利用Trowell培養(yǎng)系統(tǒng)和去除舌組織的體外器官培養(yǎng)進(jìn)行腭突的直接接觸融合實(shí)驗(yàn)，探討舌與腭發(fā)育相關(guān)性。應(yīng)用穩(wěn)定轉(zhuǎn)染W(wǎng)nt5a的C2C12細(xì)胞系體外檢測(cè)Wnt5a對(duì)骨骼肌發(fā)育的影響，CCK-8法檢測(cè)C2C12細(xì)胞增殖水平及血清饑餓法誘導(dǎo)凋亡后細(xì)胞的活性，流式細(xì)胞術(shù)Annexin V-FITC/PI雙染檢測(cè)細(xì)胞凋亡水平，誘導(dǎo)C2C12細(xì)胞肌向分化后，Real-Time PCR檢測(cè)成肌調(diào)節(jié)因子成員Myf5、MyoG和Myogenin表達(dá)變化，免疫細(xì)胞化學(xué)法檢測(cè)骨骼肌特異標(biāo)志物Myosin的表達(dá)，以探討Wnt5a對(duì)肌向分化的影響。 結(jié)果：1、利用維甲酸誘導(dǎo)腭裂模型研究頜、舌與腭的發(fā)育 （1）腭突組織基因芯片研究分析提示差異基因主要集中在轉(zhuǎn)錄調(diào)節(jié)、生長(zhǎng)調(diào)節(jié)、細(xì)胞運(yùn)動(dòng)以及細(xì)胞骨架等功能，且與WNT和SHH通路相關(guān)，尤其是GSK3β和β-TrCP等既參與WNT通路又與SHH信號(hào)通路，與腭的發(fā)育畸形相關(guān)。實(shí)時(shí)定量PCR技術(shù)證明GSK3β及β-TrCP等表達(dá)下調(diào)，F(xiàn)ZD3受體表達(dá)無(wú)明顯變化。免疫組織化學(xué)結(jié)果顯示GSK3β蛋白在E14腭突快速增殖期的腭突上皮和間充質(zhì)中均有表達(dá)，在E15.5腭裂組上頜骨發(fā)育明顯延遲，GSK3β表達(dá)在大量未分化的間充質(zhì)細(xì)胞中。在E14-14.5，腭突開始上抬至發(fā)生接觸融合的關(guān)鍵時(shí)期，實(shí)驗(yàn)組腭突仍垂直于舌的兩側(cè)，與對(duì)照組相比在麥克爾軟骨邊緣有多個(gè)TUNEL陽(yáng)性凋亡細(xì)胞。 （2）在E13.5去除舌組織并利用Trowell系統(tǒng)進(jìn)行腭器官體外培養(yǎng)3天后，實(shí)驗(yàn)組以及對(duì)照組雙側(cè)腭突都發(fā)生了融合，雙側(cè)腭突的間充質(zhì)相互貫通。在E14-16.5側(cè)腭突發(fā)育全過(guò)程中，腭裂組與對(duì)照組頦舌肌細(xì)胞核數(shù)量無(wú)明顯差異，但腭裂組肌束橫截面的總面積明顯小于對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義（P0.05），舌肌細(xì)胞增殖、凋亡水平無(wú)明顯差異，未見舌異常纖維化。舌組織基因芯片分析提示，舌發(fā)育畸形與維甲酸相關(guān)的信號(hào)網(wǎng)絡(luò)相關(guān)，DTNA，CAMK2D等表達(dá)上調(diào)2倍以上。 2、Wnt5a基因?qū)ι嗉肮趋兰“l(fā)育的影響 （1）Wnt5a在正常舌發(fā)育中的表達(dá)呈動(dòng)態(tài)變化，在E13.5舌組織中呈高表達(dá)，在E15.5舌組織中的表達(dá)明顯下調(diào)。而且在E15.5，即舌肌分化成熟期，維甲酸誘導(dǎo)腭裂組Wnt5a的表達(dá)高于對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。 （2）Wnt5a基因敲除小鼠舌體以及頦舌肌中BrdU陽(yáng)性率與正常對(duì)照組相比無(wú)顯著差異，對(duì)照組頦舌肌的舌間充質(zhì)出現(xiàn)區(qū)域性密集，實(shí)驗(yàn)組則不明顯。成肌調(diào)節(jié)因子成員Myf5和MyoD蛋白在舌內(nèi)外肌群中的表達(dá)水平均下降，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。 （3）應(yīng)用Wnt5a穩(wěn)定轉(zhuǎn)染C2C12小鼠成肌細(xì)胞系，Wnt5a基因上調(diào)后第24h、48h和72h，C2C12細(xì)胞增殖能力無(wú)明顯變化（P0.05），經(jīng)過(guò)血清饑餓24h后細(xì)胞均發(fā)生凋亡，但Wnt5a上調(diào)對(duì)細(xì)胞凋亡和活性無(wú)明顯影響（P0.05），細(xì)胞遷移也無(wú)明顯變化（P0.05）。在體外成功誘導(dǎo)C2C12細(xì)胞系肌肉方向分化后，Wnt5a基因上調(diào)可引起成肌調(diào)節(jié)因子的變化，Myf5在分化第2，3天表達(dá)下降（P0.05），MRF4在分化第2天表達(dá)明顯下降（P0.05），Myog的表達(dá)在分化第2天（P0.05）及第4天（P0.01）均下調(diào)。細(xì)胞免疫化學(xué)顯示兩組細(xì)胞都有Myosin的表達(dá)，均可形成多核肌管。 結(jié)論：過(guò)量維甲酸可導(dǎo)致以腭裂為代表的口腔頜面部多器官發(fā)育異常，但不影響腭突中嵴上皮細(xì)胞接觸后的融合。維甲酸誘導(dǎo)腭裂小鼠伴發(fā)舌發(fā)育延遲、上頜骨發(fā)育遲緩以及下頜麥克爾軟骨異常凋亡，提示了過(guò)量維甲酸可干擾頜面系統(tǒng)協(xié)同發(fā)育。 維甲酸誘導(dǎo)的腭發(fā)育異常與WNT和SHH信號(hào)通路相關(guān)，維甲酸相關(guān)信號(hào)網(wǎng)絡(luò)參與舌的發(fā)育異常。體內(nèi)敲除及體外轉(zhuǎn)染上調(diào)Wnt5a基因均可延遲骨骼肌的肌向分化，提示了Wnt5a基因具有多向調(diào)控作用。
[Abstract]:With cleft lip and palate as the representative of the maxillofacial deformity in the incidence of congenital malformation of the first development. As the disease susceptibility gene, cleft palate is caused by multiple factors and multiple gene interactions, thus causes complex, unknown mechanisms. Since most of the etiology of cleft palate who focus their abnormal the development of the palatal cleft palate reason, however, is due to a defect of palate internal defects, two is secondary to other organizations such as clinical maxillofacial, maxillary hypoplasia and mandibular retrusion, leading to abnormal tongue position, and physical barriers caused cleft palate shelves. But the other the organization of maxillofacial organs, especially as with palatal tissue structure, physiological function and growth is closely related to the molecular mechanism of tongue development, and abnormalities associated with cleft palate were rarely reported.
Retinoic acid (Retinoic acid RA) is a derivative of vitamin A, as cosmetic additives and drug treatment of skin diseases, cancer and other diseases, environmental factors become more common teratogenic. At the same time with retinoic acid pathway WNT and SHH signal pathways involved in the regulation of interaction of maxillofacial development. This study used retinoic acid induction of Wnt5a gene knockout and two kinds of maxillofacial malformation animal model, through in vivo and in vitro experiments, correlation between the molecular mechanism of abnormal development of cleft palate and tongue research as well as the two. In order to deepen our understanding of the molecular mechanism of the development of the understanding of the tongue, and may provide scientific basis for the prevention of abnormal development of cleft palate and tongue and clinical treatment.
Objective: To study the molecular mechanism of cleft palate and tongue dysplasia and the correlation between the two kinds of animal models by using retinoic acid induced and Wnt5a knockout mice to develop two kinds of animal models of maxillofacial deformities.
Methods: the Wnt5a and the establishment and application of retinoic acid inducible gene knockout mice two maxillofacial malformation model to observe histomorphological changes the phenotype of stereoscopic microscope and staining, BrdU incorporation assay to detect cell proliferation, cell apoptosis TUNEL staining detection method, Masson staining of tongue muscle fibrosis, gene microarray screening differences of cleft palate and tongue abnormal development of the real-time PCR to validate the results of gene chip, immunohistochemistry experiment to further explore the WNT signal pathways of GSK3 beta protein and expression of myogenic regulatory factor family members of Myf5 and MyoD in tongue muscle. Using Trowell culture system and the removal of tongue tissue organ culture in vitro were in direct contact with palatal fusion experiments. To investigate the correlation between tongue and palate. Effect of C2C12 cell line in vitro Wnt5a stably transfected with Wnt5a on skeletal muscle development, CCK-8 was detected by C2 The activity of cell apoptosis induced by C12 cell proliferation and the level of serum starvation, flow cytometry Annexin V-FITC/PI staining to detect cell apoptosis, C2C12 cells induced by muscle differentiation, Real-Time PCR detection of myogenic regulatory factors Myf5, expression of MyoG and Myogenin, the immune cell chemical detection of skeletal muscle specific marker expression Myosin, to discuss the effects of Wnt5a on myogenic differentiation.
Results: 1, using the retinoic acid induced cleft palate model to study the development of the jaw, the tongue and the palate.
(1) study of gene chip analysis showed that the differences of the palatal tissue mainly in gene transcription regulation, growth regulation, cell motility and cytoskeleton function, and the WNT and SHH pathway, especially GSK3 beta and beta -TrCP is involved in WNT pathway and SHH pathway, and palatine malformation related in real time. Quantitative PCR proved that GSK3 beta and beta -TrCP expression, FZD3 expression had no obvious change. The immunohistochemistry results showed that GSK3 protein were expressed in mesenchymal E14 in palatal fast proliferating of the palatal epithelium and in the E15.5 group, cleft palate maxillary growth was significantly delayed, the expression of GSK3 in a large number of undifferentiated mesenchymal cells. In E14-14.5, palatal begins to rise to the occurrence of critical period of contact fusion, on both sides of the experimental group is perpendicular to the palatal tongue, compared with the control group there were more TUNEL positive apoptotic cells in Meckel's cartilage edge.
(2) E13.5 in the removal of tongue tissue and the use of Trowell system were cultured palatal organ in vitro after 3 days, the experimental group and the control group have undergone bilateral palatal fusion, bilateral palatal mesenchymal coalescence. On the E14-16.5 side in the whole process of the development of palate, cleft palate group and control group the number of genioglossus muscle cells the obvious difference, but the total area of the cross section of the cleft palate group was significantly lower than that of control group, the difference was statistically significant (P0.05), proliferation of tongue muscle cells, no significant difference between the levels of apoptosis, no abnormal tongue tongue tissue fibrosis. Gene chip analysis showed that tongue malformation associated with retinoic acid signaling network, DTNA, CAMK2D etc. expression of more than 2 times.
2, the effect of Wnt5a gene on the development of tongue and skeletal muscle
(1) the expression of Wnt5a in normal tongue in development is dynamic, high expression of E13.5 in tongue tissues, expression of E15.5 in tongue tissue was significantly reduced. And in E15.5, namely the tongue muscle differentiation in mature stage, the expression of Wnt5a in retinoic acid induced cleft palate group than in the control group, the difference was statistically significant (P0.05).
(2) Wnt5a gene knockout mice tongue and the positive rate of BrdU in genioglossus muscle compared with normal control group had no significant difference between the control group and the genioglossus tongue mesenchymal regional intensive, the experimental group was not obvious. The expression of myogenic regulatory factors Myf5 and MyoD protein in tongue and muscles in the fall, the difference was statistically significant (P0.05).
(3) the application of Wnt5a stable transfection of C2C12 mouse myoblast cell line, Wnt5a gene up-regulated after 24h, 48h and 72h, C2C12 cell proliferation had no significant change (P0.05), after serum starvation after 24h cells underwent apoptosis, but the upregulation of Wnt5a has no obvious effect on cell apoptosis and activity (P0.05), and cell migration no significant change (P0.05). The success of induced differentiation of C2C12 cells in vitro after muscle, up-regulated Wnt5a gene can induce changes of myogenic regulatory factor, decrease the expression of Myf5 in differentiation of day 2,3 (P0.05), the expression of MRF4 decreased significantly in second days of differentiation (P0.05), the expression of Myog in differentiation (second days P0.05) and 4 days (P0.01) were down regulated. Immunocytochemistry showed that the expression of the two groups of cells is Myosin, can form multinucleated myotubes.
Conclusion: excess retinoic acid can lead to cleft palate as the representative of the oral and maxillofacial multiple organ abnormalities, but does not affect the fusion of epithelial cells in the palatal ridge after contact with the tongue. Delayed development of cleft palate in mice induced by retinoic acid, maxillary growth retardation and mandibular cartilage abnormalities Meckel apoptosis, suggesting that excess retinoic acid can be coordinated development interference and system.
Retinoic acid induced velopharyngeal dysplasia is related to WNT and SHH signaling pathway. Retinoic acid related signal network is involved in the development of tongue. In vivo knockout and in vitro transfection of Wnt5a gene can delay skeletal muscle myogenic differentiation, suggesting that Wnt5a gene has multiple regulatory effects.

【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R-332;R440;R782.22

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